1. Field of the Invention
The invention concerns staining of nucleic acids which have been fixed on a solid support and which are detected by a non-radioactive labelling method.
2. Description of the Prior Art
Detection of specific nucleic acid sequences in biological entities is becoming more and more important, particularly in detecting genetic illnesses.
Methods using solid supports are already known, for example the use of a polyamide or nitrocellulose membrane to which nucleic acids from a sample are transferred and fixed: the desired sequence may then be detected on the support.
The nucleic acid in a sample may be as natural or synthetic, modified or unmodified DNA or RNA. It is usually fragmented using restriction enzymes, the fragments subsequently being transferred and fixed onto the support by known means.
A particular sequence may then be identified using a detection system such as utilizing a "probe" which will fix itself specifically on the sequence and reveal it in situ, i.e. at the location of its fixation on the solid support.
Generally, the detection system employed comprises a sequence complementary to the sequence to be identified which can hybridize with the latter. Such hybridization only occurs when the detection system recognizes a complementary sequence held on the solid support.
Direct detection of nucleic acids is possible in certain methods without necessitating hybridization between a "target" sequence held on the solid support and a "probe" sequence forming part of the detection means.
Such nucleic acid detection, either directly or following hybridization of two complementary nucleic acid sequences, has been carried out for some time using radioactive isotopes. In methods of this type, one or more nucleosides radio-labelled with .sup.3 H or .sup.32 P are used to label the nucleic acids which are detected by counting or autoradiography. Whilst such known methods are reliable and sensitive, they unfortunately necessitate the use of laboratories specially equipped for using radioactivity. Further, such laboratories must have the required licences for handling, using and confining radioactive isotopes.
In order to overcome this drawback, detection methods which do not use radioactive isotopes have recently been developed.
One such method involves detection of nucleic acids which have previously been fixed on a solid support such as a membrane wherein an enzymatic system is employed to label the sequence non-radioactively and which is then reacted with a chromogenic substrate to form a colored product which precipitates in situ thus revealing the sequence at the reaction site.
Such detection methods are carried out using a set or "kit" of reactants containing those products necessary for characterizing specific nucleic acids.
Most commercially available nucleic acid detection kits use alkaline phosphatase as the final visual detection enzyme. Properties of this enzyme are such that non-radioactive detection methods using it are as sensitive as detection methods utilizing radioactive isotopes.
Alkaline phosphatase is particularly resistant and extremely sensitive compared with other enzymes used for this type of detection, for example peroxidase or glucosidase.
The main advantage of non-radioactive systems and methods is that they may be used diagnostically in an environment where radioactivity could not be employed for routine tests. Such new diagnostic tests, in particular ELISA tests, will replace certain known tests in all areas, particularly bacteriology, virology and parasitology. They may be used in any location where detectable nucleic acids are found, i.e. in living organisms.
The use of non-radioactive ("cold") probes represents an important advance both in fundamental and in clinical research since detection sensitivity is at least equal to that obtained with radioactive isotopes.
Non-radioactive probes are particularly advantageous since they can readily be used without requiring specialized equipment. There are no risks for the operator such as those associated with the use of radioisotopes and, further, they do not require the use of toxic or carcinogenic products.
However, even though they have several advantages over radioactive probes, cold probes do have a number of drawbacks.
Certain of these have already been overcome, as for example in detection of "unique sequences" which is now possible.
One of the major drawbacks of cold probes lies in the fact that the solid supports, e.g. membranes, cannot be used again after hybridization and colorimetric detection. This constitutes a major problem in both clinical and fundamental research.
Researchers must, in fact, reuse the supports, with the target mucleic acids irreversibly fixed thereon, several times, sometimes up to twenty times in some cases.
This is because the fixed nucleic acids may be extremely costly, whether because of their lengthy, costly and difficult extraction or because of their rarity as would be the case with foetal DNA. It is, therefore, important to be able to fix probes on the nucleic acid targets several times, for example by rehybridization.
Such rehybridization is well known with isotopic probes but impossible with cold probes because of the indelible deposit constituted by the colored stain precipitated in situ.
Several attempts have been made to enable the stain produced to be removed, so far without success. In this respect, some manufacturers have advocated the use of hot dimethylformamide to eliminate this indelible stain. Unfortunately dimethylformamide is a volatile teratogenic agent particularly when hot; this limits its use in repeated washes.
Thus, there currently exist no colorimetric detection methods using substrates which produce colored, insoluble products which may be readily eliminated after visualization so that the solid support, to which the nucleic acids to be analyzed are transferred and fixed, may be reused.
One object of the invention is a method for staining nucleic acids fixed on an appropriate solid support, which method constitutes a non-radioactive labelling detection method, the method further comprising elimination of the colored stain produced by the reaction.
Another object of the invention is such a method wherein elimination of the colored stain is obtained using products which are not dangerous to the user.
Another object of the invention is such a method which also produces a good stain and, therefore, good detection of nucleic acids, particularly for staining of unique genome sequences.
Another object of the invention is such a method which may in particular be applied to solid supports constituted by polyamide or nitrocellulose membranes to which the target nucleic acids to be analyzed are irreversibly fixed.
Another object of the invention is a method wherein the solid support, in particular a membrane, may be readily decolorized to produce a perfectly white support without any trace of staining and which may be reused immediately for a new detection operation, for example by hybridization.
Another object of the invention is a set or kit of reactants for carrying out said detection method.